Abstract
Genetically encoded peptide library screening is a powerful strategy for discovering inhibitors of protein-protein and protein-DNA interactions. The Transcription Block Survival (TBS) assay enables the in vivo selection of peptides that antagonize transcription factor (TF) function by linking the inhibition of DNA binding to E. coli survival. However, previous TBS implementations required laborious re-engineering of the mDHFR coding region for each new target, limiting utility. Here, we present an enhanced and streamlined TBS platform that increases throughput, simplifies target switching, and improves selection stringency. By relocating TF DNA-binding sites from within the mDHFR coding sequence into the mDHFR 5'-promoter/untranslated region, we preserve mDHFR folding and function, enabling rapid interchange of TF targets without the need for extensive construct redesign. We validated this system using three distinct TF targets, CREB1, ATF2, and DLX5, and two distinct consensus sites, demonstrating robust transcriptional block upon TF binding and efficient growth rescue upon peptide-mediated antagonism. Importantly, we expand the platform to accommodate full-length TFs, as exemplified by DLX5, allowing selection against biologically relevant full-length, multidomain proteins without immobilization or tags. TBS continues to function exclusively by selecting for the disruption of protein-DNA binding, ensuring mechanistic precision. Using this optimized TBS system, we successfully screened an 11.3-million-member peptide library to identify a potent antagonist of ATF2-CRE DNA binding within three months. This next generation TBS platform significantly improves screening efficiency and selection pressure while maintaining high biological relevance, providing a versatile and scalable tool for discovering functional peptide inhibitors of protein-DNA interactions with therapeutic potential.